Three-phase liquid extraction: a simple and fast method for lipidomic workflows

Abstract

This article is available online at http://www.jlr.org hydrophobic lipid species. Ceramides and diacylglycerols (DAGs) have polarities that fall in between the previous examples. This range of hydrophobicity adds complexity to the lipid analysis. For this reason, an effective method that decreases bias while isolating a representative sample of lipids from biological matrices is critical for a lipidomic analysis.Lipidomic profiling by MS has become a widely used tool in lipid biochemistry. Recent advances in MS have led to better sensitivity, improved mass accuracy, and novel methods for broad lipid profiling. ESI is commonly used in MS but is particularly sensitive to ion suppression and matrix effects. Therefore, coextraction of interferents, such as nonlipid biomolecules, leads to increased complexity of MS spectra, decreased sensitivity, and reduced lipid identification (2, 6, 7). The importance of sample preparation prior to lipidomic analysis is critical, as it can negatively influence the analytical measurements. Ensuring the quality of lipid extracts is an important step because improper sample preparation will yield questionable or misleading results. Liquid-liquid extractions (LLEs) and solid-phase extractions (SPEs) are the most common sample preparation techniques used in lipidomics and are implemented depending on analytical strategy; LLE is favored for global lipidomic analysis (8-10), while SPE is predominately used for targeted methods when the fractionation and/or isolation of a specific lipid class or classes is necessary. However, SPE can be variable, time-consuming, and challenging to automate. The efficiency of LLE depends on the partitioning of lipids into an organic phase. The most commonly Abstract An unbiased sample preparation free of interferents (i.e., competing analytes, detergents, plastics) is critical to any lipid MS workflow. Here we present a novel three-phase lipid extraction (3PLE) technique using a single-step liquid-liquid extraction (LLE) that allows both extraction and fractionation of lipids by polarity. 3PLE is composed of one aqueous and two organic phases. The upper organic phase is enriched in neutral lipids (triacylglycerols and cholesteryl esters), while the middle organic phase contains the major glycerophospholipids. Thin-layer chromatography, radioactive labeling, and MS were used to confirm lipid partitioning. 3PLE efficiency was demonstrated for bovine liver, human pooled plasma, mouse liver, mouse brain, and mouse white adipose tissue. Compared with the gold-standard Bligh/Dyer LLE, 3PLE showed significant advantages. For direct-infusion workflows, there was a decrease in ion suppression with a corresponding increased number of lipid species identified. For LC/MS workflows, increased signal intensities were observed for lower-abundance lipid species such as phosphatidic acid and phosphatidylserine. 3PLE also proved to be a valuable tool for fatty acid profiling by GC/MS, allowing for the separate identification of neutral and polar fatty acids.-Vale, G., S. A. ...

Diverse species-specific phenotypic consequences of loss of function sorting nexin 14 mutations

“…Evaporated fish water was replaced each morning between 09:00 and 09:30 a.m. Following each experiment, larvae were euthanised with an overdose of 2-Phenoxyethanol (Acros Organics), and larval DNA was extracted for genotyping using HotSHOT DNA preparation 42 .…”

“…Evaporated fish water was replaced each morning between 09:00 and 09:30 a.m. Following each experiment, larvae were euthanised with an overdose of 2-Phenoxyethanol (Acros Organics), and larval DNA was extracted for genotyping using HotSHOT DNA preparation 42 . Larval behaviour was measured by calculating the number of pixels that changed intensity within each well between each pair of frames, a metric termed Δ pixels.…”

“…Data analysis was performed by MarkerView (SCIEX) peak-picking algorithm and interrogated by an in-house script. For further details, please see full report 42 .…”

Lipidomics from sample preparation to data analysis: a primer

“…This method has comparable extraction efficiency to the Folch protocol, but can be fully automated for high-throughput screening in 96-well plates and avoids the need for hazardous chloroform. A recently published three-phase lipid extraction protocol relies on the addition of hexane, methyl acetate, acetonitrile, and water, which results in three distinct phases: a lower aqueous phase, an organic phase in the middle, and an upper organic phase [17]. While the middle organic phase contains mostly polar lipids (e.g., glycerophospholipids, sphingolipids), the upper organic phase contains mostly neutral lipids (e.g., triacylglycerols (TG)).…”

“…To deal with such uncertainty, many users have transferred from triple-quadrupole instruments to high-resolution mass spectrometers with quadrupole-time of flight (Q-TOF) or Orbitrap technology [27,29,31]. While Q-TOF-based technology offers the mass resolution of 40,000 needed to separate the above-described isobaric example of plasmalogens and diacyl PC species, the mass resolution required to separate 13 C, 17 O, 2 H, or 15 N isotopologues from either each other or other monoisotopic lipid species is above 100,000, and can only be provided by Orbitrap or ion cyclotron technology. Nevertheless, in natural samples, all minor isotopes except for 13 C can be neglected for practical purposes due to their low abundances, but it is highly beneficial to separate the M + 2 isotopic peak of a lipid, which is mainly due to 13 C, from the monoisotopic mass peak of the same lipid with one double bond less.…”